Novel Heparin Entities and Methods of Use

ABSTRACT

The present invention relates to immobilized biologically active entities that retain a significant biological activity following manipulation. The invention also comprises a medical substrate comprising a heparin entity bound onto a substrate via at least one heparin molecule, wherein said bound heparin entity is heparinase-1 sensitive.

FIELD OF THE INVENTION

The present invention relates to medical substrates having immobilizedbiologically active entities that maintain their biological activityafter sterilization. Specifically the present invention relates to newheparin entities and their method of use.

BACKGROUND OF THE INVENTION

Medical devices which serve as substitute blood vessels, synthetic andintraocular lenses, electrodes, catheters and the like in and on thebody or as extracorporeal devices intended to be connected to the bodyto assist in surgery or dialysis are well known. However, the use ofbiomaterials in medical devices can stimulate adverse body responses,including rapid thrombogenic action. Various plasma proteins play a rolein initiating platelet and fibrin deposition on biomaterial surfaces.These actions lead to vascular constriction that hinder blood flow, andthe inflammatory reaction that follows can lead to the loss of functionof the medical device. Biologically active entities that reduce orinhibit thrombus formation on the surface of a biomaterial and/orcovering material are of particular interest for blood contactingdevices. Glycosaminoglycans are generally preferred anti-thromboticagents; with heparin, heparin analogs, and derivatives beingparticularly preferred.

Immobilization of glycosaminoglycans, such as heparin, to biomaterialshas been researched extensively to improve bio- and hemocompatibility.The mechanism responsible for reducing thrombogenicity of a heparinizedmaterial is believed to reside in the ability of heparin to speed up theinactivation of serine proteases (blood coagulation enzymes) byanti-thrombin III (ATIII). In the process, ATIII forms a complex with awell defined pentasaccharide sequence in heparin, undergoing aconformational change and thus enhancing the ability of ATIII to form acovalent bond with the active sites of serine proteases, such asthrombin. The formed serine protease-ATIII complex is then released fromthe heparin, leaving said heparin behind for subsequent rounds ofinactivation via a catalytic process.

Immobilization of biologically active entities, such as heparin, onbiomaterials in a biologically active form involves an appreciation ofthe respective chemistries of the entity and the biomaterial. In thefield of medical devices, ceramic, polymeric, and/or metallic materialsare common biomaterials. These materials can be used for implantabledevices, diagnostic devices or extracorporeal devices. Modification ofthe chemical composition of a biomaterial is often required toimmobilize a biologically active entity thereon. This modification isusually accomplished by treating surfaces of the biomaterial to generatea population of chemically reactive moieties or groups, followed byimmobilization of the biologically active entity with an appropriateprotocol. With other biomaterials, surfaces of a biomaterial arecovered, or coated, with a material having reactive chemical groupsincorporated therein. Biologically active entities are then immobilizedon the biomaterial through the reactive chemical groups of the coveringmaterial. A variety of schemes for covering, or coating, biomaterialshave been described. Representative examples of biologically activeentities immobilized to a biomaterial with a covering, or coating, aredescribed in U.S. Pat. Nos. 4,810,784; 5,213,898; 5,897,955; 5,914,182;5,916,585; and 6,461,665.

When biologically active compounds, compositions, or entities areimmobilized, the biological activity of these “biologics” can benegatively impacted by the process of immobilization. The biologicalactivity of many biologics is dependent on the conformation andstructure (i.e., primary, secondary, tertiary, etc.) of the biologic inits immobilized state. In addition to a carefully selectedimmobilization process, chemical alterations to the biologic may berequired for the biologic to be incorporated into the covering materialwith a conformation and structure that renders the biologic sufficientlyactive to perform its intended function.

Despite an optimized covering and immobilization scheme, additionalprocessing, such as sterilization, can degrade the biological activityof the immobilized biologic. For implantable medical devices,sterilization is required prior to use. Sterilization may also berequired for in vitro diagnostic devices having sensitivity tocontaminants. Sterilization of such devices often requires exposure ofthe devices to elevated temperature, pressure, and humidity, often forseveral cycles. In some instances, antimicrobial sterilants, such asethylene oxide gas (EtO) or vapor hydrogen peroxide, are included in thesterilization process. In addition to sterilization, mechanicalcompaction and expansion, or long-term storage of an immobilizedbiologic can degrade the activity of the biologic.

There exists a need for medical devices having biologically activeentities immobilized thereon that can be subjected to sterilization,mechanical compaction and expansion, and/or storage without significantloss of biological activity. Such a medical device would havebiologically compatible compositions or compounds included with theimmobilized biological entities that serve to minimize degradation ofthe biological activity of the entities during sterilization, mechanicalcompaction and expansion, and/or storage. In some instances, theadditional biologically compatible compositions or compounds wouldincrease the biological activity of some biologically active entitiesfollowing a sterilization procedure.

SUMMARY OF THE INVENTION

Thus, the present invention comprises medical substrates comprisingheparin entities immobilized onto a substrate. The heparin entities ofthe invention retain significant biological activity followingimmobilization, sterilization, mechanical compaction and expansion,and/or storage, as compared to other coated medical substrates.

One embodiment of the invention comprises a medical substrate comprisinga heparin entity bound onto a substrate via at least one heparinmolecule, wherein said bound heparin entity is heparinase sensitive. Inanother embodiment, said substrate is selected from the group consistingof polyethylene, polyurethane, silicone, polyamide-containing polymers,polypropylene, polytetrafluoroethylene,expanded-polytetrafluoroethylene, fluoropolymers, polyolefins, ceramics,and biocompatible metals. In another embodiment, said substrate isexpanded-polytetrafluoroethylene. In another embodiment, saidbiocompatible metal is a nickel-titanium alloy, such as Nitinol. Inanother embodiment, said substrate is a component of a medical device.In another embodiment, said medical device is selected from the groupconsisting of grafts, vascular grafts, stents, stent-grafts, bifurcatedgrafts, bifurcated stents, bifurcated stent-grafts, patches, plugs, drugdelivery devices, catheters, cardiac pacemaker leads, balloons, andindwelling vascular filters. In another embodiment, after heparinasetreatment, heparin, or fragments thereof, will not be detected on saidsubstrate.

Another embodiment of the invention comprises a heparin entitycomprising at least one heparin molecule and at least one core moleculesuch that when said heparin entity is bound onto a substrate via a leastone heparin molecule, said heparin entity is heparinase sensitive. Inone embodiment, said core molecule is selected from the group consistingof proteins (including polypeptides), hydrocarbons, aminoglycosides,polysaccharides and polymers. In another embodiment, said heparin entityis bound onto a substrate via at least one heparin molecule and whereinsaid bound heparin molecule is attached to said substrate via end pointattachment. In another embodiment, said heparin entity is bound onto asubstrate via at least one heparin molecule and wherein said boundheparin molecule is attached to said substrate via loop attachment.

Another embodiment of the invention comprises an ATIII binding entitycomprising a core molecule, at least one polysaccharide chain attachedto the core molecule, and at least one free terminal aldehyde moiety onthe polysaccharide chain. In one embodiment, said polysaccharide chainis heparin or a heparin fragment. In another embodiment, said coremolecule is selected from the group consisting of a protein (includingpolypeptides), a hydrocarbon, an aminoglycoside, a polysaccharide and apolymer. In another embodiment, said substrate is selected from thegroup consisting of polyethylene, polyurethane, silicone,polyamide-containing polymers, polypropylene, polytetrafluoroethylene,expanded-polytetrafluoroethylene, fluoropolymers, polyolefins, ceramics,and biocompatible metals. In another embodiment, said ATIII bindingentity is bound onto a substrate via end-point attachment or loopattachment. In another embodiment, said substrate is a component of amedical device.

Another embodiment of the invention comprises a method of determiningthe structure of a heparin entity bonded to a substrate, comprising thesteps of providing a substrate comprising a heparin entity,depolymerizing the heparin entity to generate a mixture of solubleheparin fragments, detecting each soluble heparin fragment in saidmixture using column chromatography, determining the identity of eachdetected heparin fragment from the above step, and deriving thestructure of the heparin entity from the identities of the detectedheparin fragments. In one embodiment, said depolymerizing is byheparinase depolymerization. In another embodiment, said columnchromatography is strong anion exchange high performance liquidchromatography (SAX-HPLC).

Another embodiment of the invention comprises a system for determiningthe structure of a heparin entity bonded to a substrate, comprising adepolymerization solution, a labeling reagent solution, and a detector.In another embodiment, said depolymerization solution comprisesheparinase. In another embodiment, said labeling reagent solutioncomprises toluidine blue and terbium tris(4-methylthio)benzoate. Inanother embodiment, said detector comprises SAX-HPLC, an epifluoroscentmicroscope, and an absorption spectroscope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts several heparin entities of the invention and types ofattachment of said heparin entities to a substrate.

FIG. 2 depicts ATIII binding capacity of various aldehyde containingheparin entities conjugated onto expanded polytetrafluoroethylene(ePTFE) and having undergone multiple EtO sterilizations. Aldehydecontaining heparin entities are classified according to the coremolecule used in the synthesis of the heparin entity. Hence, colistinsulfate as the core refers to Examples 1, neomycin to Example 2,poly-L-lysine to Example 4, capreomycin to Example 3, polyethyleneimine(PEI) to Example 5, and ethylene diamine (EDA) to Example 6. All barsrepresent mean values of samples numbers with error bars for thestandard deviation.

FIGS. 3A and B depict light micrographs of heparin entities comprisingfree terminal aldehydes immobilized onto an ePTFE substrate by a singleend-point attachment method before (3A) and after (3B) treatment withheparinase-1 and stained with toluidine blue. The absence of colorationin Figure B as compared to A, demonstrates that heparin entitiescomprising free terminal aldehydes immobilized onto an ePTFE substrateby a single end-point attachment method is essentially depolymerizedfrom the surface after heparinase-1 treatment.

FIG. 3C depicts the normalized change in luminosity before and aftertreatment with heparinase-1 for heparin immobilized through end-pointaldehyde and multi-point attachment, heparin entities comprising aneomycin core immobilized through end-point and multi-point attachmentthrough at least one heparin molecule, and USP heparin immobilizedthrough multi-point attachment. The low normalized change in luminosityvalues for the heparin end-point aldehyde, heparin entity comprisingheparin and neomycin core with end-point aldehyde, and USP heparin, allmulti-point attached to the substrate, indicated that the surfaces arenot heparinase-1 sensitive and still have substantial heparin on thesurface.

FIGS. 4A-C depicts light micrographs of heparin entities comprisingheparin and an EDA core immobilized onto an ePTFE substrate by a singleend-point attachment method before (4A and 4B) and after (4C) treatmentwith heparinase-1 and stained with toluidine blue. The stained samplesdemonstrate the presence of the heparin entity. Samples 4B and 4C weresubjected to a round of sterilization and rinsed only with DI water poststerilization. The coloration of FIG. 4C after sterilization andheparinase-1 treatment indicates that heparinase-1 did not recognizeheparin entities on the surface.

FIGS. 4D and E depict light micrographs of heparin entities comprisingheparin and an EDA core immobilized onto an ePTFE substrate by a singleend-point attachment method before (4D) and after (4E) treatment withheparinase-1 and stained with toluidine blue. These samples wheresubjected to a round of sterilization and rinsed with DI water and boricacid post sterilization. The lack coloration of FIG. 4E aftersterilization indicates that heparinase-1 did recognize heparin entitieson the surface and depolymerized them.

FIGS. 5A-C depicts SAX-HPLC chromatograms from heparinase-1depolymerization of (A) USP heparin, (B) heparin entities constructedfrom heparin and colistin sulfate, and (C) heparin entities constructedfrom heparin and neomycin sulfate.

FIGS. 6A and B depicts SAX-HPLC chromatograms from heparinase-1depolymerization of ePTFE surface immobilized (a) USP heparin bound byfree terminal aldehyde and (b) heparin entities constructed from heparinand colistin sulfate bound by free terminal aldehyde.

DETAILED DESCRIPTION

The present invention comprises medical substrates comprising heparinentities immobilized onto a substrate. The heparin entities of theinvention retain significant biological activity followingimmobilization and sterilization as compared to other coated medicalsubstrates.

In the context of this disclosure, a number of terms are used. Thefollowing definitions are provided. As used herein and in the appendedclaims, the singular forms “a”, “an”, and “the” include plural referenceunless the context clearly dictates otherwise.

As used herein the term “heparin entity” means heparin moleculescovalently attached to a core molecule. Said heparin molecules can beattached to the core molecule by end point attachment (as describedbelow and as essentially described in U.S. Pat. No. 4,613,665,incorporated by reference herein for all purposes) or other methodsknown in the art (see e.g. G T Hermanson, Bioconjugate Techniques,Academic Press, 1996; H G Garg et al., Chemistry and Biology of Heparinand Heparan Sulfate, Elsevier, 2005.)

As used herein the term “core molecule” means a polyfunctional moleculeto which heparin is attached. For the purposes of this invention, saidcore molecule and a substrate are not the same, although a core moleculeand a substrate can be made from the same material.

As used herein, the term “substantially pure” means, an object speciesis the predominant species present (i.e., on a molar basis it is moreabundant than any other individual species in the composition), andpreferably a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to about 90 percent ofall macromolecular species present in the composition. Most preferably,the object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of a singlemacromolecular species.

As used herein, the term “heparinase” means any enzymatic reaction thatdepolymerizes (e.g. digests) heparin. Examples of heparinase include,but are not limited to, heparinase-1, heparinase-2, heparinase-3,heparanase, exosulphatases, bacterial exoenzymes, and glycosidases thatcan depolymerize heparin.

As used herein the term “heparinase sensitive” means that aftertreatment of a substrate comprising heparin entities with heparinase andstaining said substrate with toluidine blue, the substrate will not bevisibly stained (essentially as depicted in FIG. 3B and FIG. 4E). Theterm also means that an insignificant amount of toluidine blue will bindto residual heparin, or fragments thereof, and a reading from a detectorthat can measure the amount of toluidine blue (or other labels) on asubstrate, such as a spectrophotometer, luminometer, densitometer,liquid scintillation counter, gamma counter, or the like, will be aboutbackground levels, or be insignificantly different from backgroundlevels when compared to a substrate without heparin entities and stainedwith toluidine blue, or be below the sensitivity of said detectors whencompared to a substrate comprising heparin entities and stained withtoluidine blue without heparinase treatment. The term also means that alabel that binds to heparin, or fragments thereof, will not detect asubstantial amount of heparin, or fragments thereof, after treatment ofa substrate comprising heparin entities with heparinase.

As used herein the terms “bound,” “attached,” and “conjugate,” and theirderivatives, when referring to heparin entities and/or heparin meanscovalently bound, unless specified otherwise.

Referring to FIGS. 1A-C, one embodiment of the invention comprises amedical substrate comprising a heparin entity 100 bound onto a substrate106 via at least one heparin molecule 104, wherein said bound heparinentity is heparinase sensitive. Suitable substrate materials forimmobilizing or binding said heparin entities comprise polymers such as,but not limited to, polyamides, polycarbonates, polyesters, polyolefins,polystyrene, polyurethane, poly(ether urethane), polyvinyl chlorides,silicones, polyethylenes, polypropylenes, polyisoprenes,polytetrafluoroethylenes, and expanded-polytetrafluoroethylenes (ePTFE,as described in U.S. Pat. No. 4,187,390). In one embodiment, expanded,or porous, polytetrafluoroethylene (ePTFE) is the substrate.

Additional substrates include, but are not limited to, hydrophobicsubstrates such as polytetrafluoroethylene, expandedpolytetrafluoroethylene, porous polytetrafluoroethylene, fluorinatedethylene propylene, hexafluoropropylene, polyethylene, polypropylene,nylon, polyethyleneterephthalate, polyurethane, rubber, silicone rubber,polystyrene, polysulfone, polyester, polyhydroxyacids, polycarbonate,polyimide, polyamide, polyamino acids, regenerated cellulose, andproteins, such as silk, wool, and leather. Methods of making porouspolytetrafluoroethylene materials are described in U.S. Pat. Nos.3,953,566 and 4,187,390, each of which is incorporated herein byreference. In another embodiment, said ePTFE may be impregnated, filled,imbibed or coated with at least one chemical compound known to cause abioactive response. Compounds that cause a bioactive response compriseanti-microbials (e.g. anti-bacterials and anti-virals),anti-inflammatories (e.g. dexamethasone and prednisone),anti-proliferatives (e.g. taxol, paclitaxel and docetaxel) andanti-coagulating agents (e.g. abciximab, eptifibatide and tirofibran).In one embodiment, said anti-inflammatory is a steroid. In anotherembodiment, said steroid is dexamethasone. Methods of coating substratesare well known in the art. In another embodiment, said substratecomprises the heparin entities of the invention and a coating thatcomprises a compound that causes a bioactive response. Said substratecomprises the materials referred to above and below. In one embodiment,said substrate is ePTFE.

Other suitable substrates include, but are not limited to, cellulosics,agarose, alginate, polyhydroxyethylmethacrylate, polyvinyl pyrrolidone,polyvinyl alcohol, polyallylamine, polyallylalcohol, polyacrylamide, andpolyacrylic acid.

Additionally, certain metals and ceramics may be used as substrates forthe present invention. Suitable metals include, but are not limited to,titanium, stainless steel, gold, silver, rhodium, zinc, platinum,rubidium, and copper, for example. Suitable alloys includecobalt-chromium alloys such as L-605, MP35N, Elgiloy, nickel-chromiumalloys (such as Nitinol), and niobium alloys, such as Nb-1% Zr, andothers.

Suitable materials for ceramic substrates include, but are not limitedto, silicone oxides, aluminum oxides, alumina, silica,hydroxyapapitites, glasses, calcium oxides, polysilanols, andphosphorous oxide. In another embodiment, protein-based substrates, suchas collagen can be used. In another embodiment, polysaccharide-basedsubstrates, such as cellulose can be used.

Some substrates may have multiplicities of reactive chemical groupspopulating at least a portion of its surface to which heparin entitiesof the invention can be bound. Said heparin entities of the inventionare covalently bound to the substrate material through said reactivechemical groups. Surfaces of said substrates can be smooth, rough,porous, curved, planar, angular, irregular, or combinations thereof. Insome embodiments, substrates with surface pores have internal voidspaces extending from the porous surface of the material into the bodyof the material. These porous substrates have internal substratematerial bounding the pores that often provides surfaces amenable toimmobilizing biologically active entities. Whether porous or non-porous,substrates can be in the form of filaments, films, sheets, tubes,meshworks, wovens, non-wovens, and combinations thereof.

Substrates lacking reactive chemical groups on their surfaces (orlacking appropriately reactive chemical groups) can be covered, at leastin part, with a polymeric covering material having a multiplicity ofreactive chemical groups thereon to which said heparin entities can bebound. Polymeric substrates can also be modified along their surface, oralong their polymer backbone using a variety of methods, includinghydrolysis, aminolysis, photolysis, etching, plasma modification, plasmapolymerization, carbene insertion, nitrene insertion, etc. Said heparinentities are covalently attached, or bound, to the polymeric coveringmaterial through the reactive chemical groups of the covering materialor directly to a substrate that has been modified. The polymericcovering material may form at least one layer on at least a portion of asubstrate.

There are many other surface modifications, such those described U.S.Pat. No. 4,600,652 and U.S. Pat. No. 6,642,242, which are based onsubstrates having a layer of a polyurethane urea to which heparinmodified to contain aldehyde groups through oxidation with nitrous acidor periodate, may be bound by covalent links. A similar technology isdescribed in U.S. Pat. No. 5,032,666, where the substrate surface iscoated with an amine rich fluorinated polyurethane urea beforeimmobilization of an antithrombogenic agent, such as analdehyde-activated heparin. Another antithrombogenic surfacemodification which may be mentioned is described in publicationWO87/07156. The surface of the device is modified through the coatingwith a layer of lysozyme or a derivative thereof to which heparin isadhered. Yet another surface modification for producing antithrombogenicarticles is described in U.S. Pat. No. 4,326,532. In this case, thelayered antithrombogenic surface comprises a polymeric substrate, achitosan bonded to the polymeric substrate and an antithrombogenic agentbonded to the chitosan coating. Others have reported an antithrombogenichemofilter also using a chitosan layer for binding heparin. Anotherprocess for preparing antithrombogenic surfaces is described inWO97/07834, wherein the heparin is admixed with sufficient periodate soas not to react with more than two sugar units per heparin molecule.This mixture is added to a surface modified substrate of a medicaldevice, wherein said surface modification contains amino groups. Theabove listing of processes for adding reactive groups to substrates areonly a small example of how this can be accomplished. The above listingis by no means complete. Furthermore, it is clear that the type ofprocess used to add reactive chemical groups to a substrate will dependon the properties of the substrate of which a person of skill in the artwill recognize.

In another embodiment of the invention, said medical substratecomprising said bound heparin entity via at least one heparin moleculeis a component of a medical device. Medical devices comprise, but arenot limited to, grafts, vascular grafts, stents, stent-grafts,bifurcated grafts, bifurcated stents, bifurcated stent-grafts, herniapatches, hernia plugs, periodontal grafts, surgical fabrics, drugdelivery devices, catheters, cardiac leads balloons and indwellingfilters. In one embodiment, said stents can be used in cardiac,peripheral or neurological applications. In another embodiment, saidstent-grafts can be used in cardiac, peripheral or neurologicalapplications.

Another embodiment of the invention comprises a heparin entitycomprising at least one heparin molecule and at least one core molecule.As shown in FIG. 1, the core molecule 102 is the “backbone” of theheparin entity 100 to which heparin molecules 104 are bound. Said coremolecule 102 can be either cyclic (102 a, FIGS. 1A and 1C), linear (102b, FIG. 1B), branched, dendritic, “Y” shaped, “T” shaped, or “star”shaped as described by Freudenberg, U., Biomaterials, 30, 5049-5060,2009 and Yamaguchi, N., Biomacromolecules, 6, 1921-1930, 2005. In oneembodiment, said core molecule is selected from the group consisting ofproteins (including polypeptides), hydrocarbons, lipids,aminoglycosides, polysaccharides and polymers. Proteins include, but arenot limited to, antibodies, enzymes, receptors, growth factors,hormones, serpins and any globular protein. Specific proteins andpolypeptides include, but are not limited to, albumin, colistin,collagen, polylysine, antithrombin III, fibrin, fibrinogen, thrombin,laminin, keratin, and the like. In another embodiment, said coremolecule can be a polypeptide. Said polypeptide need not be very longand can comprise one or more repetitions of amino acids, for examplerepetitions of serine, glycine (e.g. Ser-Gly-Gly-Ser-Gly), lysine orornithine residues. Alternatively, other amino acid sequences can beused, for example colistin, polylysine, and polymyxin.

Examples of polysaccharides include, but are not limited to neutralpolysaccharides such as cellulose, starch, agarose,carboxymethylcellulose, nitrocellulose, and dextran, anionicpolysaccharides such as alginate, heparin, heparin sulfate, dextransulfate, xanthan, hyaluronic acid, carrageenan, gum arabic, tragacanth,arabinogalactan, and pectin; macrocyclic polysaccharides such ascyclodextrin and hydroxypropyl cyclodextrin; and polycationicpolysaccharides such as chitin and chitosan.

Examples of synthetic polymers include, but are not limited to,polyethylene glycol (PEG) 200, 300, 400, 600, 1000, 1450, 3350, 4000,6000, 8000 and 20000, polytetrafluoroethylene, polypropylene glycol,poly(ethylene glycol-co-propylene glycol), copolymers of polyethyleneglycol, copolymers of polypropylene glycol, copolymers oftetrafluoroethylene with vinyl acetate and vinyl alcohol, copolymers ofethylene with vinyl acetate & vinyl alcohol, polyvinyl alcohol,polyethyleneimine, polyacrylic acid; polyols such as polyvinyl alcoholand polyallyl alcohol; polyanions such as acrylic acid andpoly(methacrylic acid). Polycation polymers include poly(allylamine),poly(ethyleneimine), poly(guanidine), poly(vinyl amine), polyethyleneglycol diamine, ethylene diamine, and poly(quaternary amines);polyacrylonitriles such as hydrolyzed polyacrylonitrile,poly(acrylamide-co-acrylonitrile), and their copolymers. Other polymersinclude fluorinated copolymers including copolymers oftetrafluoroethylene and vinyl alcohol, vinyl acetate, vinyl formamide,acrylamide, and vinyl amine. In another embodiment, said core moleculecan be an aminoglycoside, including, but not limited to, amikacin,arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin, and apramycin.

Heparin is a mucopolysaccharide, isolated from pig intestine or bovinelung and is heterogeneous with respect to molecular size and chemicalstructure. Heparin is built up from alternating glycuronic acid andglucosamine units. The glycuronic acid units consist of D-glycuronicacid and L-iduronic acid. These are respectively D- and L-(1,4)-bound tothe D-glucosamine units. A large proportion of the L-iduronic acidresidues are sulfated in the 2-position. The D-glucosamine units areN-sulfated, sulfated in the 6-position and are α-(1,4)-bound to theuronic acid residues. Certain D-glucosamine units are also sulfated inthe 3-position. Heparin contains material with a molecular weightranging from about 6,000 Daltons to about 30,000 Daltons. The hydroxyland amine groups are derivatized to varying degrees by sulfation andacetylation. The active sequence in heparin responsible for itsanticoagulation properties is a unique pentasaccharide sequence thatbinds to the ligand anti-thrombin III (ATIII). The sequence consists ofthree D-glucosamine and two uronic acid residues. Heparin molecules canalso be classified on the basis of their pentasaccharide content. Aboutone third of heparin contains chains with one copy of the uniquepentasaccharide sequence (see, Choay, Seminars in Thrombosis andHemostasis 11:81-85 (1985) which is incorporated herein by reference)with high affinity for ATIII, whereas a much smaller proportion(estimated at about 1% of total heparin) consists of chains whichcontain more than one copy of the high affinity pentasaccharide (see,Rosenberg et al., Biochem. Biophys. Res. Comm. 86:1319-1324 (1979) whichis incorporated herein by reference). The remainder (approx. 66%) of theheparin does not contain the pentasaccharide sequence. Thus, so called“standard heparin” constitutes a mixture of the three species: “highaffinity” heparin is enriched for species containing at least one copyof the pentasaccharide and “very high affinity” heparin refers to theapproximately 1% of molecules that contain more than one copy of thepentasaccharide sequence. These three species can be separated from eachother using routine chromatographic methods, such as chromatography overan anti-thrombin affinity column (e.g., Sepharose-AT; see, e.g., Lam etal., Biochem. Biophys. Res. Comm. 69:570-577 (1976) and Homer Biochem.J. 262:953-958 (1989) which are incorporated herein by reference).

In one embodiment, said heparin is derived from an animal. In anotherembodiment, said heparin is bovine or porcine derived. In anotherembodiment, said heparin is a synthetic heparin, i.e. not derived fromanimal sources (e.g. fondaparinux or enoxaparin). In another embodiment,heparin entities of the invention comprise heparin that has beenenriched and comprises substantially pure “high affinity” heparin. Inanother embodiment, heparin entities of the invention comprise heparinthat has been enriched and comprises substantially pure “very highaffinity” heparin. In another embodiment, heparin entities of theinvention comprises heparin has been enriched and comprises acombination of substantially pure “high affinity” and “very highaffinity” heparin.

Another embodiment of the invention comprises the binding of saidheparin entity to a medical substrate via at least one heparin molecule.As shown in FIG. 1, the heparin entities of the invention are bound tosaid substrate via at least one heparin molecule. Thus, in oneembodiment, said bound heparin molecule is attached to said substratevia end point attachment (as depicted in FIGS. 1A and 1B). In anotherembodiment, said bound heparin molecule is attached to said substratevia an end point aldehyde. This can be accomplished essentially asdescribed in U.S. Pat. No. 4,613,665, which is incorporated herein byreference in its entirety, and as described below.

In another embodiment, said heparin entity is bound onto a substrate viaat least one heparin molecule, wherein said bound heparin molecule isattached to said substrate via a “loop attachment.” Loop attachment, asdepicted in FIG. 1C, is an attachment of said heparin entity via atleast one heparin, wherein the heparin is attached loosely to thesubstrate in a small number of locations, therefore allowing substantialportions of the bound heparin to be exposed to heparinase (as opposed tomore common methods that attach heparin tightly in a large number oflocations). The more common methods of coupling heparin to a substratecomprise reacting a majority of functional groups randomly localizedalong a heparin molecule's length (e.g. using coupling agents such ascarbodiimides, epoxides, and polyaldehydes). These methods result in ahigh probability that the active sequence (said unique pentasaccharidesequence describe above) will be bound to the substrate resulting inreduced and/or lost activity. In loop attachment of heparin, only a fewfunctional groups on the heparin react and are bound to the substrate.Thus, there is a high probability that the active sequence of theattached heparin will not be bound to the substrate, therefore allowingsaid active sequence to bind to its ligand. In another embodiment, theinvention comprises a heparin entity with multiple attachments to asubstrate, wherein the active sequence is not bound to the substrate. Inanother embodiment, said bound heparin entity molecule is attached tosaid substrate via loop attachment.

As discussed above, endpoint and loop attachments allow a substantialportion of at least one heparin molecule (in a heparin entity) not to bebound to a substrate. As used herein the term “substantial portion”means that about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 96% about 97%, about 98% and about 99% of the heparinmolecule is not bound to the substrate. In another embodiment, the termalso refers to the at least one heparin molecule (in a heparin entity)wherein said the at least one heparin molecule bound to the substrate isnot bound to a substrate via its active sequence. Thus, since the activesequence is not bound to the surface of the substrate, the activesequence has a greater probability of interacting with its ligand. Inother words, if the active sequence is bound to the surface of thesubstrate then there is a small chance of heparin binding to its ligand.

However, because said heparin entities are attached via heparin byendpoint and/or loop attachment, the heparin is sensitive to heparinase.Thus, after heparinase treatment, there will be very little, if any,heparin, or fragments thereof, on the surface of said substrate. Incontrast, some of the more common methods of attaching heparin to thesurface of a substrate (which comprises multiple bonds along the lengthof the heparin molecule, as described above), after heparinasetreatment, will have a significant amount of heparin, or fragmentsthereof, still attached to the surface of the substrate. Thus, afterheparinase treatment, heparin, or fragments thereof, can be detected onthe surface of said substrate. Without being bound to any particulartheory, the inventors have that discovered that the more sensitive thebound heparin or heparin entity is to heparinase, the more biologicalactivity said bound heparin or heparin entity exhibits. This may bebecause the active sequence of the bound heparin or heparin entity isnot attached to the surface of the substrate, thus said bound heparin orheparin entity has a greater chance of binding to its ligand.

Heparin must have intact conformation and structure to be recognized byATIII, and if said conformation and structure is lost, heparin willexhibit poor activity. In addition, loss of said conformation andstructure results in poor recognition by other proteins, such asheparinase-1, resulting in said heparin being resistant todepolymerization. For example, modification of soluble heparin withcarbodiimide changes the soluble heparin structure in such a way that itis no longer recognized by heparinase-1, and the modified solubleheparin has reduced whole blood anticoagulant activity (see Olivera, G.B., Biomaterials, 24, 4777-4783, 2003). The inventors have discoveredthat heparinase sensitivity of attached heparin or heparin entity ispredictive of ATIII binding activity of said attached heparin or heparinentity. Without wanting to be constrained by any particular theory, ifthe attached heparin or heparin entity retains specificity for specificenzymes such as heparinase-1, then the attached heparin or heparinentity retains substantially enough primary/secondary/tertiary structurefor it also to have specificity for ATIII. Thus, the inventors havediscovered that when an attached heparin or heparin entity is recognizedby heparinase-1, said attached heparin or heparin entity is alsorecognized by ATIII, as exemplified by high binding activities.

The inventors have also shown that a boric acid rinse will restoreheparinase sensitivity to inactivated attached heparin or heparinentities (inactivated by sterilization, mechanical compaction andexpansion, or long-term storage, for example). Thus, another embodimentof the invention comprises a method of restoring heparinase sensitivityto heparin or heparin entities bound onto a substrate comprising rinsingsaid substrate in a solution of boric acid. In one embodiment, saidsubstrate was exposed to a sterilization cycle. In another embodiment,said substrate was exposed to mechanical treatments that reducedheparinase-1 activity.

In another embodiment of the invention, after treating a medicalsubstrate with bound heparin entities of the invention with heparinase,heparin, or fragments thereof, will not be detected on said substrate.In another embodiment, after treating a medical substrate with boundheparin entities of the invention with heparinase, heparin, or fragmentsthereof, will be detected at a substantially lower level than beforeheparinase treatment. Significantly lower level of detection comprisesvery little detection after staining and/or labeling for heparin.

In another embodiment, said heparin, or fragments thereof, will not bedetected visually (macroscopically) after staining or labeling. Heparin,or fragments thereof, can be detected by a label that binds directly orindirectly to heparin, or fragments thereof. In one embodiment, saidlabel that binds to heparin, or fragments thereof, is selected from thegroup consisting of dyes, antibodies, and proteins. Examples of labelsinclude, but are not limited to proteins including anti-heparinantibodies (polyclonal or monoclonal) and ATIII; metachromatic dyesincluding toluidine blue, azure A, alcian blue, victoria blue 4R, nightblue, methylene blue; radioiodinated labels including radioiodinatedtoluidine blue, radioiodinated methylene blue, radioiodinated heparinantibodies, radioiodinated ATIII; tritiated labels including tritiatedtoluidine blue, tritiated azure A, tritiated alcian blue, tritiatedvictoria blue 4R, tritiated night blue, tritiated methylene blue;carbon-14 labels including 14C-toluidine blue, 140-azure A, 14C-alcianblue, 140-victoria blue 4R, 140-night blue, 14C-methylene blue;fluorescent labels including rhodamine-labelled heparin antibodies,fluorescein-labelled heparin antibodies, rhodamine-labelled ATIII,fluorescein-labelled ATIII. In another embodiment, said dye is toluidineblue. In another embodiment, after heparinase treatment, aninsignificant amount of toluidine blue will bind to heparin, orfragments thereof, but will not be visually detected on said substrate(essentially as depicted in FIGS. 3B and 4E). In another embodiment,after heparinase treatment, a insignificant amount of toluidine bluewill bind to residual heparin, or fragments thereof, and a reading froma detector that can measure the amount of toluidine blue (or otherlabels described above) on a substrate (e.g. a spectrophotometer,luminometer, densitometer, liquid scintillation counter, gamma counter,or the like) will be about background levels, or be insignificantlydifferent from background levels when compared to a substrate withoutheparin entities. In another embodiment, after heparinase treatment, areading from a detector that can measure the amount of toluidine blue(or other labels described above) on a substrate will be significantlydifferent when compared to a substrate comprising heparin entities andstained with toluidine blue (or other labels described above) withoutheparinase treatment.

Another embodiment of the invention comprises a heparin entitycomprising at least one heparin molecule attached to a core molecule,wherein the entity is bound to a substrate via a heparin molecule, andwherein after exposure to heparinase and toluidine blue, the substratemacroscopically evidences substantially no toluidine blue on its surface(as depicted in FIG. 3B and FIG. 4E).

Another embodiment of the invention comprises a heparin entity whichcomprises at least one heparin molecule and at least one core moleculesuch that when said heparin entity is bound onto a substrate via a leastone heparin molecule, said heparin entity is heparinase sensitive. Inone embodiment, said substrate is selected from the group consisting ofpolyethylene, polyurethane, silicone, polyamide-containing polymers,polypropylene, polytetrafluoroethylene,expanded-polytetrafluoroethylene, biocompatible metals, ceramics,proteins, polysaccharides, and any substrate described above. In anotherembodiment, said substrate is expanded-polytetrafluoroethylene. Inanother embodiment, said substrate is a component of a medical device.In another embodiment, said medical device is selected from the groupconsisting of grafts, vascular grafts, stents, stent-grafts, bifurcatedgrafts, bifurcated stents, bifurcated stent-grafts, patches, plugs, drugdelivery devices, catheters and cardiac leads. In another embodiment,said stents can be used in cardiac, peripheral or neurologicalapplications. In another embodiment, said stent-grafts can be used incardiac, peripheral or neurological applications. In another embodiment,said medical device can be used in orthopedic, dermal, or gynecologicapplications. In another embodiment, said core molecule comprises acyclic, linear, branched, dendritic, “Y”, “T”, or star molecularstructure. In another embodiment, said core molecule is selected fromthe group consisting of proteins, polypeptides, hydrocarbons,polysaccharides, aminoglycosides, polymers, and fluoropolymers.

In another embodiment, heparin, or fragments thereof, is detected bylabels that bind to heparin, or fragments thereof. In anotherembodiment, said label that binds to heparin, or fragments thereof, isselected from the group consisting of dyes, polyclonal antibodies, andproteins. In another embodiment, said dye is toluidine blue. In anotherembodiment, after heparinase treatment, an insignificant amount oftoluidine blue will bind to residual heparin, or fragments thereof, andwill not be visually detected on said substrate. In another embodiment,after heparinase treatment, a insignificant amount of toluidine bluewill bind to residual heparin, or fragments thereof, and a reading froma detector that can measure the amount of toluidine blue (or otherlabels described above) on a substrate (e.g. a spectrophotometer,luminometer, densitometer, liquid scintillation counter, gamma counter,or the like) will be about background levels, or be insignificantlydifferent from background levels when compared to a substrate withoutheparin entities. In another embodiment, after heparinase treatment, areading from a detector that can measure the amount of toluidine blue(or other labels described above) on a substrate will be significantlydifferent when compared to a substrate comprising heparin entities andstained with toluidine blue (or other labels described above) withoutheparinase treatment. In another embodiment, said heparin entity isbound onto a substrate via at least one heparin molecule and whereinsaid bound heparin molecule is attached to said substrate via end-pointattachment. In another embodiment, said heparin entity is bound onto asubstrate via at least one heparin molecule, wherein said bound heparinmolecule is attached to said substrate via end-point aldehyde. Inanother embodiment, said heparin entity is bound onto a substrate via atleast one heparin molecule, wherein said bound heparin molecule isattached to said substrate via loop attachment. In another embodiment,said heparin entity is bound onto a substrate via at least one heparinmolecule, wherein said bound heparin molecule is attached to saidsubstrate via aldehydes along the length said heparin.

Another embodiment of the invention comprises an ATIII binding entitycomprising: a core molecule, a polysaccharide chain attached to the coremolecule, and a free terminal aldehyde moiety on the polysaccharidechain. This ATIII binding entity can then be end-point attached to asubstrate via a terminal aldehyde. Another embodiment of the inventioncomprises an ATIII binding entity comprising: a core molecule, apolysaccharide chain attached to the core molecule, and free terminalaldehyde moieties along the length of the polysaccharide chain. ThisATIII binding entity can then be looped attached to a substrate via thealdehydes along the length of the polysaccharide chain. In anotherembodiment, said polysaccharide chain is heparin. In another embodiment,said core molecule is selected from the group consisting of a protein, apolypeptide, a hydrocarbon, an aminoglycoside, a polysaccharide, apolymer, a fluoropolymer, or any core molecule described herein. Inanother embodiment, heparin is bound onto the core molecule viaend-point attachment. In another embodiment, the substrate is selectedfrom the group consisting of polyethylene, polyurethane, silicone,polyamide-containing polymers, and polypropylene,polytetrafluoroethylene, expanded-polytetrafluoroethylene andbiocompatible metals, or any of the substrates described herein. Inanother embodiment said biocompatible metal is Nitinol. In anotherembodiment, said substrate is expanded-polytetrafluoroethylene. Inanother embodiment, said substrate is a component of a medical device.In another embodiment, said medical device is selected from the groupconsisting of grafts, vascular grafts, stents, stent-grafts, bifurcatedgrafts, bifurcated stents, bifurcated stent-grafts, patches, plugs, drugdelivery devices, catheters and cardiac leads. In another embodiment,said medical device can be used in cardiac, peripheral, neurologic,orthopedic, gynecologic, or dermal applications.

Another embodiment of the invention comprises an implantable medicaldevice comprising a medical substrate, wherein said medical substratecomprises a heparin entity bound onto a substrate via at least oneheparin molecule, wherein said bound heparin entities are heparinasesensitive. In one embodiment, said medical device is selected from thegroup consisting of grafts, vascular grafts, stents, stent-grafts,bifurcated grafts, bifurcated stents, bifurcated stent-grafts, patches,plugs, drug delivery devices, catheters and cardiac leads. In anotherembodiment, said stent can be used in cardiac, peripheral orneurological applications. In another embodiment, said stent can be aballoon expandable and/or a self expanded stent. Said stents can be madefrom any biocompatible material including any polymer or metal asdescribed above. In another embodiment, said stent is made from Nitinoland/or stainless steel. In another embodiment, said stent comprises agraft. In another embodiment, said graft and/or stent comprise heparinentities of the invention.

The heparin entities of the invention retain significant biologicalactivity following immobilization and sterilization as compared to othercoated medical substrates. Thus, in one embodiment said medicalsubstrate comprises, a heparin entity bound onto a substrate via atleast one heparin molecule, wherein said bound heparin entity isheparinase sensitive has an ATIII activity of about 300 pmol/cm². Inanother embodiment; the ATIII activity is about 250 pmol/cm², about 200pmol/cm², about 150 pmol/cm², about 100 pmol/cm², about 50 pmol/cm²,about 40 pmol/cm², about 30 pmol/cm², about 20 pmol/cm², about 10pmol/cm² or about 5 pmol/cm². In another embodiment, after a first roundof sterilization the ATIII activity of said medical substrate is about250 pmol/cm², about 200 pmol/cm², about 150 pmol/cm², about 100pmol/cm², about 50 pmol/cm², about 40 pmol/cm², about 30 pmol/cm², about20 pmol/cm², about 10 pmol/cm² or about 5 pmol/cm². In anotherembodiment, after a second round of sterilization, the ATIII activity ofsaid medical substrate is about 100 pmol/cm², about 90 pmol/cm², about80 pmol/cm², about 70 pmol/cm², about 60 pmol/cm², about 50 pmol/cm²,about 40 pmol/cm², about 30 pmol/cm², about 20 pmol/cm², about 10pmol/cm² or about 5 pmol/cm². In another embodiment, after a third roundof sterilization, the ATIII activity of said medical substrate is aboveabout 50 pmol/cm², or about 70 pmol/cm², about 60 pmol/cm², about 50pmol/cm², about 40 pmol/cm², about 30 pmol/cm², about 20 pmol/cm², about10 pmol/cm² or about 5 pmol/cm². ATIII activity assays are well known inthe art and at least one is described below. In another embodiment, saidheparin entities of the invention retain significant biological activityfollowing compression and expansion of a medical device. In anotherembodiment, said heparin entities of the invention retain significantbiological activity following storage conditions for medical deviceseither in a compacted and/or expanded state.

Another embodiment of the invention comprises methods of determining thestructure of a heparin entity bonded to a substrate. One method ofdetermining the structure of a heparin entity bonded to a substratecomprises the steps of: providing a substrate comprising a heparinentity, depolymerizing the heparin entity to generate a mixture ofsoluble heparin fragments, detecting each soluble heparin fragment insaid mixture using column chromatography, determining the identity ofeach detected soluble heparin fragment from above, and deriving thestructure of the heparin entity from the identities of the detectedsoluble heparin fragments. In one embodiment, said depolymerization isby heparinase-1. In another embodiment, column chromatography is stronganion exchange-high performance liquid chromatography or SAX-HPLC.

Another embodiment of the invention comprises an implantable medicaldevice comprising a medical substrate, wherein said medical substratecomprises a heparin entity bound onto a substrate via at least oneheparin molecule, wherein said bound heparin entities are heparinasesensitive. In one embodiment, said medical device is selected from thegroup consisting of grafts, vascular grafts, stents, stent-grafts,bifurcated grafts, bifurcated stents, bifurcated stent-grafts, patches,plugs, drug delivery devices, catheters, cardiac leads, balloons andindwelling filters. In another embodiment, said stent can be used incardiac, peripheral or neurological applications. In another embodiment,said stent can be a balloon expandable and/or a self expanded stent.Said stents can be made from any biocompatible material including anypolymer or metal as described above. In another embodiment, said stentis made from Nitinol and/or stainless steel. In another embodiment, saidstent comprises a graft. In another embodiment, said graft and/or stentcomprise heparin entities of the invention.

Another embodiment of the invention comprises methods of determining thespatial distribution of a heparin entity bonded to a substrate. Onemethod of determining the spatial distribution of a heparin entitybonded to a substrate comprises the steps of: providing a substratecomprising a heparin entity, depolymerizing the heparin entity togenerate a surface comprising surface-bonded unsaturated heparinfragments, reacting the surface with a labeling reagent which introducesa detectable component to said surface-bonded unsaturated heparinfragments, detecting said surface-bonded unsaturated heparin fragmentvia said detectable component, and deriving the spatial distribution ofthe heparin entity from the presence of the surface-bonded unsaturatedheparin fragments. In one embodiment, depolymerization is byheparinase-1. In another embodiment, said labeling reagent is alanthanoid Michael-like addition organo-complex. In another embodiment,said labeling reagent is terbium tris(4-methylthio)benzoate. In anotherembodiment, said organo-complex comprises chemisorbed goldnanoparticles. In another embodiment, said detecting is byepifluoroscent microscopy or transmission electron microscopy.

Another embodiment of the invention comprises a system for determiningthe structure of a heparin entity bonded to a substrate, comprising adepolymerization solution, a labeling reagent solution, and a detector.A system is an assembly of reagents and instruments used to detect thestructure and type of binding of heparin entities to a substrate. In oneembodiment, said depolymerization solution comprises heparinase-1. Inanother embodiment, said labeling reagent solution comprises toluidineblue, and terbium tris(4-methylthio)benzoate. In another embodiment,said detector comprises SAX-HPLC, an epifluoroscent microscope, and anabsorption spectroscope. In another embodiment, said assembly ofreagents can be a kit.

After enzymatic heparinase-1 depolymerization of heparin and/or heparinentities that are end-point attached, heparin fragments are left are onthe surface that are unsaturated, i.e. they comprise a carbon-carbondouble bond (“nubs”). Enzymatic heparinase depolymerization involvescleavage of the non-reducing terminal uronic acid residue to a4,5-unsaturated derivative. This produces residual surface-bondedunsaturated heparin fragments bonded to the substrate that comprises acarbon-carbon double bond. Thus, the structure of the residualsurface-bonded heparin fragment is unsaturated, and can react withvarious detection molecules, including those that comprise Michael-likeaddition complexes, such as thiol-containing compounds andthiol-containing fluorescent compounds, such as terbiumtris(4-methylthio)benzoate. Thus, in another embodiment of theinvention, after enzymatic heparinase-1 depolymerization of an end-pointattached heparin entity, said residual surface-bonded unsaturatedheparin fragments bonded to the substrate comprising a carbon-carbondouble bond are detected. This method can determine if heparin and/orheparin entities were end-point attached to a substrate. In anotherembodiment, nub detection is combined with any of the detection and/orcharacterization methods described above.

This invention is further illustrated by the following Examples whichshould not be construed as limiting. The contents of all Figures andreferences are incorporated herein by reference.

EXAMPLES Example 1

This example describes the construction of heparin entities comprisingheparin and colistin sulfate as the core. This heparin entity containsfree terminal aldehydes that can be used for attachment to a surface ofa substrate.

Colistin sulfate (0.10 g, Alpharma, Inc.) was dissolved in 300 ml ofdeionized (DI) water containing MES buffer (pH 4.7, BupH™ ThermoScientific). To this was added 10 g USP heparin, 4 gN-hydroxysulfosuccinimide (sulfo-NHS, Thermo Scientific), and 4 g of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC hydrochloride,Sigma-Aldrich, St. Louis, Mo.). The reaction was allowed to proceed atroom temperature for 4 hours, followed by dialysis overnight with a50,000 MWCO membrane (Spectra/Por®). The retentate (about 350 ml out of500 ml) was transferred to a beaker, and cooled to 0° C. Sodium nitrite(10 mg) and acetic acid (2 ml) were added and the reaction was allowedto proceed for 1 hour at 0° C. Dialysis was performed overnight with a50,000 MWCO membrane with the addition of 1 g NaCl to the dialysisliquid. Freezing and lyophilization of the retentate produced a finepowder.

Example 2

This example describes the construction of heparin entities comprisingheparin and neomycin sulfate as the core. This heparin entity containsfree terminal aldehydes that can be used for attachment to a surface ofa substrate.

Neomycin sulfate (0.0646 g, Spectrum Chemical) was dissolved in 300 mlof DI water containing MES buffer (pH 4.7, BupH™ Thermo Scientific). Tothis was added 10 g USP heparin, 4 g N-hydroxysulfosuccinimide(sulfo-NHS), and 4 g of EDC hydrochloride. The reaction was allowed toproceed at room temperature for 4 hours, followed by dialysis overnightwith a 50,000 MWCO membrane (Spectra/Por®). The retentate (about 400 mlout of 505 ml) was transferred to a beaker and cooled to 0° C. Sodiumnitrite (10 mg) and acetic acid (2 ml) were added and the reaction wasallowed to proceed for 1 hour at 0° C. Dialysis was performed overnightwith a 50,000 MWCO membrane with the addition of 1 g NaCl to thedialysis liquid. The dialyzed retentate was filtered twice using a 20micrometer, 0.00079 inches U.S.A. standard testing sieve, A.S.T.M.E.-11specification NO. 635 to remove small particles. Freezing of thefiltrate and lyophilization produced a fine powder.

Example 3

This example describes the construction of heparin entities comprisingheparin and capreomycin sulfate as the core. This heparin entitycontains free terminal aldehydes that can be used for attachment to asurface of a substrate.

Capreomycin sulfate (0.0501 g, Sigma-Aldrich, St. Louis, Mo.) wasdissolved in 300 ml of DI water containing MES buffer (pH 4.7, BupH™Thermo Scientific). To this was added 10 g USP heparin, 4 gN-hydroxysulfosuccinimide (sulfo-NHS), and 4 g of EDC hydrochloride. Thereaction was allowed to proceed at room temperature for 4 hours. Thereaction mixture was filtered once using a 20 micrometer, 0.00079 inchesU.S.A. standard testing sieve, A.S.T.M.E.-11 specification NO. 635 toremove small particles and the filtrate was dialyzed overnight with a50,000 MWCO membrane (Spectra/Por®). The retentate (about 400 ml out of515 ml) was transferred to a beaker and cooled to 0° C. Sodium nitrite(10 mg) and acetic acid (2 ml) were added and the reaction was allowedto proceed for 1 hour at 0° C. Dialysis was performed overnight with a50,000 MWCO membrane with the addition of 1 g NaCl to the dialysisliquid. The retentate was filtered twice using a 20 micrometer, 0.00079inches U.S.A. standard testing sieve, A.S.T.M.E.-11 specification NO.635 to remove small particles. Freezing of the filtrate andlyophilization produced a fine powder.

Example 4

This example describes the construction of heparin entities comprisingheparin and poly-L-lysine as the core. This heparin entity contains freeterminal aldehydes that can be used for attachment to a surface of asubstrate.

Poly-L-lysine (0.1776 g, Sigma-Aldrich, molecular weight 1,000 to 5,000g/mole) was dissolved in 300 ml of DI water containing MES buffer (pH4.7, BupH™ Thermo Scientific). To this was added 10 g USP heparin, 4 gN-hydroxysulfosuccinimide (sulfo-NHS), and 4 g of EDC hydrochloride. Thereaction was allowed to proceed at room temperature for 4 hours followedby dialysis overnight with a 50,000 MWCO membrane (Spectra/Por®). Theretentate (about 400 ml out of 505 ml) was transferred to a beaker andcooled to 0° C. Sodium nitrite (10 mg) and acetic acid (2 ml) were addedand the reaction was allowed to proceed for 1 hour at 0° C. Dialysis wasperformed overnight with a 50,000 MWCO membrane with the addition of 1 gNaCl to the dialysis liquid. Freezing of the retentate andlyophilization produced a fine powder.

Example 5

This example describes the construction of heparin entities comprisingheparin and polyethyleneimine (PEI) as the core. This heparin entitycontains free terminal aldehydes that can be used for attachment to asurface of a substrate.

PEI (Lupasol, BASF, 1.7756 g) was dissolved in 300 ml of DI watercontaining MES buffer (pH 4.7, BupH™ Thermo Scientific). To this wasadded 10 g USP heparin, 4 g N-hydroxysulfosuccinimide (sulfo-NHS), and 4g of EDC hydrochloride. The reaction was allowed to proceed at roomtemperature for 4 hours followed by dialysis overnight with a 50,000MWCO membrane (Spectra/Por®). The retentate (about 400 ml out of 505 ml)was transferred to a beaker and cooled to 0° C. Sodium nitrite (10 mg)and acetic acid (2 ml) were added and the reaction was allowed toproceed for 1 hour at 0° C. Dialysis was performed overnight with a50,000 MWCO membrane with the addition of 1 g NaCl to the dialysisliquid. Freezing of the retentate and lyophilization produced a finepowder.

Example 6

This example describes the construction of heparin entities comprisingheparin and ethylene diamine (EDA) as the core. This heparin entitycontains free terminal aldehydes that can be used for attachment to asurface of a substrate.

EDA (0.0043 g, Sigma-Aldrich, St. Louis, Mo.) was neutralized to a pH of4.7 with equal volume dilution of HCl and DI water, with the use of anice bath, then dissolved in 300 ml of DI water containing MES buffer (pH4.7, BupH™ Thermo Scientific). To this was added 10 g USP heparin, 4 gN-hydroxysulfosuccinimide (sulfo-NHS), and 4 g of EDC hydrochloride. Thereaction was allowed to proceed at room temperature for 4 hours followedby dialysis overnight with a 50,000 MWCO membrane (Spectra/Por®). Theretentate (about 400 ml out of 505 ml) was transferred to a beaker andcooled to 0° C. Sodium nitrite (10 mg) and acetic acid (2 ml) were addedand the reaction was allowed to proceed for 1 hour at 0° C. Dialysis wasperformed overnight with a 50,000 MWCO membrane with the addition of 1 gNaCl to the dialysis liquid. Freezing of the retentate andlyophilization produced a fine powder.

Example 7

The heparin entities containing free terminal aldehydes of Examples 1through 6 were immobilized onto the surface of an ePTFE substrate andtested for ATIII activity.

An ePTFE substrate material in sheet form was obtained from W.L. Gore &Associates, Inc., Flagstaff, Ariz. under the trade name GORE™Microfiltration Media (GMM-406). A covering material in the form of abase coating was applied to the ePTFE material by mounting the materialon a ten centimeter (10 cm) diameter plastic embroidery hoop andimmersing the supported ePTFE material first in 100% isopropyl alcohol(IPA) for about five minutes (5 min) and then in a solution ofpolyethylene imine (PEI, Lupasol, BASF) and IPA in a one to one ratio(1:1). LUPASOL® water-free PEI was obtained from BASF and diluted to aconcentration of about four percent (4%) and adjusted to pH 9.6.Following immersion of the ePTFE material in the solution for aboutfifteen minutes (15 min), the material was removed from the solution andrinsed in DI water at pH 9.6 for 15 min. PEI remaining on the ePTFEmaterial was cross-linked with a 0.05% aqueous solution ofglutaraldehyde (Amresco) at pH 9.6 for 15 min. Additional PEI was addedto the construction by placing the construction in a 0.5% aqueoussolution of PEI at pH 9.6 for 15 min and rinsing again in DI water at pH9.6 for 15 min. The imine formed as a result of the reaction betweenglutaraldehyde and the PEI layer is reduced with a sodiumcyanborohydride (NaCNBH₃) solution (5 g dissolved in 1 L DI water, pH9.6) for 15 min and rinsed in DI water for thirty minutes (30 min).

An additional layer of PEI was added to the construction by immersingthe construction in 0.05% aqueous glutaraldehyde solution at pH 9.6 for15 min, followed by immersion in a 0.5% aqueous solution of PEI at pH9.6 for 15 min. The construction was then rinsed in DI water at pH 9.6for 15 min. The resultant imines were reduced by immersing theconstruction in a solution of NaCNBH₃ (5 g dissolved in 1 L DI water, pH9.6) for 15 min followed by a rinse in DI water for 30 min. A thirdlayer was applied to the construction by repeating these steps. Theresult was a porous hydrophobic fluoropolymeric base material, or diskhaving a hydrophilic cross-linked polymer base coat on substantially allof the exposed and interstitial surfaces of the base material.

An intermediate chemical layer was attached to the polymer base coat inpreparation for placement of another layer of PEI on the construction.The intermediate ionic charge layer was made by incubating theconstruction in a solution of dextran sulfate (Amersham PharmaciaBiotech) and sodium chloride (0.15 g dextran sulfate and 100 g NaCldissolved in 1 L DI water, pH 3) at 60° C. for ninety minutes (90 min)followed by rinsing in DI water for 15 min.

A layer of PEI, referred to herein as a “capping layer” was attached tothe intermediate layer by placing the construction in a 0.3% aqueoussolution of PEI (pH 9) for about forty-five minutes (45 min) followed bya rinse in a sodium chloride solution (50 g NaCl dissolved in 1 L DIwater) for twenty minutes (20 min). A final DI water rinse was conductedfor 20 min.

The heparin entities containing free terminal aldehydes of Examples 1through 6 were attached, or conjugated, to the PEI layer(s) by placingthe construction in a heparin entity-containing sodium chloride saltsolution (approximately 0.9 g of heparin entity containing free terminalaldehydes, 5.88 g NaCl dissolved in 200 ml DI water, pH 3.9) and keptfor ten minutes (10 min) at 60° C. A 572 μL volume of a 2.5% (w/v)aqueous NaCNBH₃ solution was added to the (200 ml) heparin entitysolution. Samples were kept for additional one hundred ten minutes (110min) at the above temperature.

The samples were then rinsed in DI water for 15 min, borate buffersolution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1 LDI water, pH 9.0) for 20 min, and finally in DI water for 15 minfollowed by lyophilization of the entire construction to produce a dryconstruct comprising a heparin entity bound to the surface of the ePTFEsubstrate material. The presence and uniformity of the macromolecularconstruct of heparin was determined by staining samples of theconstruction on both sides with toluidine blue. The staining produced anevenly stained surface indicating heparin was present and uniformlybound to the ePTFE material.

Samples approximately one square centimeter (1 cm²) in nominal size werecut from the construction and assayed for heparin activity by measuringthe ATIII binding capacity of the heparin entities containing freeterminal aldehydes that were end-point attached onto the surface of theePTFE substrate. The assay is described by Larsen M. L., et al., in“Assay of plasma heparin using thrombin and the chromogenic substrateH-D-Phe-Pip-Arg-pNA (S-2238).” Thromb Res 13:285-288 (1978) and PascheB., et al., in “A binding of antithrombin to immobilized heparin undervarying flow conditions.” Artif. Organs 15:281-491 (1991), both of whichare incorporated by reference herein for all purposes. The results wereexpressed as amount of ATIII bound per unit surface area substratematerial in picomoles per square centimeter (pmol/cm²). All samples weremaintained in a wet condition throughout the assay. It is important tonote that while the approximately one square centimeter (1 cm²) sampleseach have a total surface area of two square centimeters (2 cm²) if bothsides of the material are considered, only one surface on the sample(i.e., 1 cm²) was used for calculating ATIII heparin entity-bindingactivity in pmol/cm².

Lyophilized samples representing each conjugated constructs produced inExamples 1 through 6 were placed in an individual Tower DUALPEEL® SelfSealing Pouch (Allegiance Healthcare Corp., McGraw Park, Ill.) andsealed for EtO sterilization. Ethylene oxide sterilization was carriedout under conditions of conditioning for one hour (1 hr), an EtO gasdwell time of 1 hr, a set point temperature of 55° C., and an aerationtime of twelve hours (12 hrs). Sterilization with EtO was repeated up to3 times with samples taken after each EtO sterilization.

FIG. 2 is a bar graph illustrating the ATIII binding capacity of heparinentities containing free terminal aldehydes from Examples 1 through 6immobilized onto an ePTFE surface and having undergone up to three EtOsterilization cycles. Anti-thrombin III binding activity is expressed aspicomoles of bound anti-thrombin III per square centimeter of substratematerial. As seen from the results, all conjugated heparin entitiescontaining free terminal aldehydes resulted in high anti-thrombin IIIbinding activity before sterilization and following up to three EtOsterilizations. All bars represent mean values of sample numbers witherror bars for the standard deviation.

Example 8

The heparin entities containing free terminal aldehydes produced inExamples 2, 3, 4, and 6 were analyzed in order to determine theirabsolute molecular weights.

A Waters 2414 RI detector in conjunction with Wyatt ASTRA 5.3.4.10software was used to determine the dn/dc for USP heparin in 100 mM NaNO₃with 0.02% NaN₃ at a laser wavelength of 660 nm. The dn/dc (change inrefractive index divided by change in concentration) for USP heparin wasdetermined by plotting known concentrations of heparin versus the RIdetector response and calculating the slope.

The heparin entities were analyzed with a Wyatt-Dawn Helleos-II 18-anglelight scattering detector (Wyatt Technology Corp.) for measurement ofabsolute molecular weight, with detectors 1, 2, 3, 4, 17, and 18 notutilized. A stock solution of the heparin entity was prepared in 100 mMNaNO₃ with 0.02% NaN₃ mobile phase. From this stock solution thefollowing concentrations were made: 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0mg/mL, and 2.5 mg/mL for the heparin entities of Examples 3 and 4. Forthe heparin entities of Examples 2 and 6, concentrations of 0.25 mg/mL,0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, and 2.0 mg/mL were made. Each samplewas filtered with a 0.02 micron syringe filter using a 5 ml syringeprior to injection into the light scattering detector. Batch dataanalysis was performed on all samples using a Zimm plot and the do/dcfor USP heparin (0.126 L/g). Table 1 depicts the absolute molecularweights.

TABLE 1 Absolute Molecular Weight Values for Heparin Entities CoreExample # Molecule Mw (g/mol) 2 Neomycin 18,570 3 Capreomycin 17,710 4Poly-L-Lysine 20,300 6 EDA 21,850 USP Heparin 14,810

All heparin entities analyzed for absolute molecular weight showedvalues larger than USP heparin (14,810 g/mol), with the values rangingfrom 17,710 g/mol for the heparin entity comprising heparin andcapreomycin as the core, to 21,850 g/mol for the heparin entitycomprising heparin and EDA as the core.

Example 9

This example demonstrates a detection method for discerning the methodof attachment of heparin or a heparin entity onto a surface of asubstrate. Specifically, this example looks at attachment of heparin andheparin entities via immobilization onto an ePTFE substrate, using asingle point attachment comprising free-terminal aldehydes, and using amulti-point attachment comprising carbodiimide conjugation.

Heparin end-point aldehyde was made according to U.S. Pat. No. 4,613,665and immobilized onto PEI-ePTFE substrates as described in Example 7.This produced a surface in which the heparin was immobilized byend-point attachment. Heparin attachment was demonstrated by staining asample with toluidine blue and noting the coloration, as shown in FIG.3A.

A surface was also produced in which the heparin end-point aldehyde wasattached not by the free terminal aldehyde, but by multiple carboxylicacid residues along the heparin chain length using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). EDC conjugation ofheparin onto a surface is known to bind the heparin through amultiplicity of sites. A PEI containing disk of Example 7 was immersedinto 300 ml of 0.1 MES buffer (pH 4.7). To this solution, 1 gram ofheparin with end-point aldehydes and 4 grams EDC hydrochloride wasadded. The reaction was allowed to proceed at room temperature for 4hours. The immobilized heparin disk was rinsed with DI water, boratebuffer, and a final DI water rinse.

A surface was also produced in which USP heparin containing no freeterminal aldehydes was attached by EDC through multiple bond sites onthe surface. A PEI containing disks of Example 7 was immersed into 300ml of 0.1 MES buffer (pH 4.7). To the solution, 1 gram USP heparin and 4grams EDC hydrochloride was added. The reaction was allowed to proceedat room temperature for 4 hours. The immobilized heparin disk was rinsedwith DI-water, borate buffer, and a final DI water rinse.

The heparin entity of Example 2 immobilized onto ePTFE/PEI as describedin Example 7 was also produced. Alternatively, the heparin entity ofExample 2 was immobilized onto ePTFE/PEI using carbodiimide conjugation.A PEI containing disk of Example 7 was immersed into 300 ml of 0.1 MESbuffer (pH 4.7). To the solution, 1 gram of the heparin entity ofExample 2 and 4 grams EDC hydrochloride was added. The reaction wasallowed to proceed at room temperature for 4 hours. The immobilizedheparin entity disk was rinsed with DI water, borate buffer, and a finalDI water rinse.

To demonstrate that heparin and heparin entities were immobilized byeach technique described above, samples approximately 1×1 cm werestained with toluidine blue. It was noted that all samples stained withtoluidine blue similar to FIG. 3A (which depicts USP heparin end-pointaldehyde immobilized by end-point attachment).

For each of the various ePTFE-PEI disks conjugated with heparin andheparin entity, a 2×2 cm square was cut and placed in a 1.5 ml vial. Tothis was added 1 ml of heparinase-1 (from Flavobacterium heparinum, E.C.4.2.2.7, Sigma-Aldrich, St. Louis, Mo.) diluted to 1 mg/mL in thefollowing buffer: 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 4 mM CaCl2, 0.01%BSA. The sample was incubated for 30 minute at room temperature, rinsedwith DI-water, and stained with toluidine blue. Samples that whereend-point attached, and not multi-point attached, appeared substantiallyless stained, as shown in FIG. 3B for USP heparin end-point aldehydeimmobilized by end-point attachment. Multi-point immobilized heparinentities retained stain.

Quantitation of the staining was performed utilizing luminositymeasurements for each of the samples. Samples were mounted onto glassslides and secured with a single strip of adhesive tape. Digital imageswere taken with an Olympus SZX12 microscope (Olympus America Inc.)equipped with an Olympus DP71 digital camera controlled with DPController 3.1.1.267 software. Images were captured using a 1× lens at7× magnification with exposure set to 1/350 sec and lighted with anoverhead ring-light. Before capture of final images, images wereexamined to ensure saturation was not exceeded. It is important to notethat stained samples, i.e., those that stained substantially withtoluidine blue, produced low luminosity values, while those that did notstain substantially produced high luminosity values (the luminosityscale for this example ranged from 0 to 255).

The luminosity of each captured digital image was assessed using AdobePhotoshop Elements 2.0 (Adobe Systems Inc., San Jose, Calif.). WithinAdobe Photoshop Elements 2.0 the image was loaded (resolution of 144pixels/inch) and a representative rectangular region of the sample wasoutlined using the rectangular marquee tool. From the top tool bar,image was selected followed by selection of histogram. The histogramwindow opened, with the channel set to luminosity. The mean is recordedas the mean luminosity.

All samples conjugated with heparin and heparin entity (tabulated withluminosity values in Table 2) stained substantially with toluidine blue,indicating dense coverage of attached heparin and heparin entity on thesubstrate. Luminosity values after immobilization and staining rangedfrom 27.3 for heparin end-point aldehyde immobilized by end-pointattachment to 139.1 for USP heparin immobilized by multi-pointattachment with EDC. After heparinase-1 treatment and staining,luminosity values increased for all samples, indicating a loss ofheparin and a consequential decrease in staining and in coloration. Forsamples more sensitive to heparinase-1, the change is more significant.This change is demonstrated in a graph of normalized change inluminosity. The term “normalized change in luminosity” is defined as theluminosity value after immobilization subtracted from the luminosityvalue after heparinase-1 treatment divided by the luminosity afterimmobilization value, with the resultant multiplied by 100, i.e.,{[(luminosity(post heparinase)−luminosity(preheparinase)]=luminosity(pre heparinase)}*100. Normalized change inluminosity for each of the samples in Table 2 is shown in FIG. 3C,displayed as a function of heparin entity type and immobilizationattachment method. The normalized change in luminosity of heparinend-point aldehyde was dependent upon the immobilization attachmentmethod, with end-point attachment giving a value of 603 and multi-pointattachment giving a value of 66. This dependency was observed for theheparin entity of heparin and neomycin with an end-point attachmentvalue of 231 and multi-point attachment of 16. USP heparin withmulti-point attachment also exhibited a low normalized change inluminosity with a value of 14. Low values in normalized change inluminosity indicated a surface resistant to heparinase-1 and hence smallquantities of heparin removed.

The heparinase-1 was effective at removing the heparin or heparin entityfrom the surface, as indicated by a lack of substantial staining bytoluidine blue and a consequential lack of coloration, and aconsequential high value for normalized change in luminosity.Heparinase-1 was utilized to discern whether heparin or a heparin entitywas attached via free terminal aldehydes or via multi-point attachmentusing carbodiimide conjugation.

TABLE 2 Luminosity Values Luminosity after Luminosity afterImmobilization Immobilization & Heparinase-1 Heparin Entity MethodStaining & Staining Heparin end- End-point 27.3 192.3 point aldehydeHeparin end- EDC multi- 73.6 122.8 point aldehyde point USP heparin EDCmulti- 139.1 158.9 point Heparin and EDC multi- 82.8 96.2 neomycin corepoint Example 2 Heparin and End-point 57.0 189.3 neomycin core Example 2

Example 10

This example demonstrates a detection method for discerning the methodof attachment of a heparin entity onto a surface of a substrate aftersterilization. Specifically, this example looks at attachment of heparinentities via immobilization onto an ePTFE substrate, using a singlepoint attachment comprising free-terminal aldehydes followed bysterilization and a boric acid rinse.

The heparin entity of Example 6 was immobilized onto ePTFE as describedin Example 7. Samples where shown to have good heparin coverage asindicated by toluidine blue staining (as shown in FIG. 4A). Otherend-point attached samples were sterilized, as described in Example 7,via 3 cycles of EtO, as described in Example 7. A portion of thesesamples was rinsed in DI water for 15 min, borate buffer solution (10.6g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1 L DI water, pH9.0) for 20 min, and finally in DI water for 15 min after sterilizationand before toluidine blue staining and measuring luminosity as describedin Example 9.

Samples having undergone sterilization but no boric acid rinse stainedwith toluidine blue before heparinase-1 treatment (FIG. 4B) and after(FIG. 4C). Both samples indicate the presence of heparin entity bycoloration and low luminosity values of 31.7 and 85.6, respectively.Sterilization has appeared to diminish the ability of heparinase-1 todepolymerize the heparin entity bound by the free terminal aldehyde, ascompared to heparin entity that was not sterilized.

Samples having undergone sterilization and boric acid rinse were stainedwith toluidine blue before heparinase-1 treatment (FIG. 4D) and after(FIG. 4E). Dense heparin entity coverage was indicated beforeheparinase-1 treatment by toluidine blue stain and a luminosity value of54.4, while the sample receiving the boric acid rinse and heparinase-1had essentially no toluidine blue stain and a luminosity value of 186.3,indicating substantial heparinase sensitivity of attached heparinentity.

This example shows that boric acid restored heparin conformation of theattached heparin entity, exemplified by high ATIII specificity andheparinase sensitivity. Without wishing to be bound by theory, it ishypothesized sterilization altered the conformation of the immobilizedheparin entity layer, substantially reducing specificity for ATIII (asevidenced by low activity) and reducing heparinase sensitivity (asevidenced by substantial staining with toluidine blue). It is furtherhypothesized the boric acid rinse restored conformation to the attachedheparin entity layer that was altered by sterilization. Restoration ofconformation resulted in sensitivity of the attached heparin entity toheparinase-1 depolymerization, as shown by lack of staining in FIG. 4E.It is further hypothesized that if an attached heparin entity has aconformation that heparinase-1 recognizes, then ATIII will recognize theattached heparin entity, and visa versa.

Example 11

This example demonstrates a detection method for determining thecomposition of the heparin entities using oligosaccharide mapping ofheparinase-1 depolymerized heparin entities with strong anionexchange-high performance liquid chromatography (SAX-HPLC).

USP Heparin and the heparin entities of Examples 1 and 2 were dissolvedat 0.1 mg in 100 μl of 50 mM acetate buffer, pH 7.3, containing 2.5 mmolof calcium acetate. The USP heparin and the heparin entities of Examples1 and 2 were depolymerized to their constituent oligosaccharides by theaddition of 6 milliunits of heparinase-1 for 15 hrs at 30° C., and flashfrozen at -85° C.

Analysis of the oligosaccharides from each sample were performed bySAX-HPLC and quantified at 232 nm using a 5 micron SAX column (150×4.6mm; Spherisorb, Waters). Isocratic separation was performed from 0 to 5min with 50 mM NaCl, pH 4.0, and linear gradient separation wasperformed from 5 to 90 min with 100% 50 mM NaCl, pH 4.0, to 100% 1.2 MNaCl, pH 4.0, at a flow of 1.2 mL/min.

FIG. 5 shows qualitative maps of (A) depolymerized USP heparin, (B) thedepolymerized heparin entity of Example 1 comprising heparin and a corecomprising colistin sulfate, and (C) the depolymerized heparin entity ofExample 2 comprising heparin and a core comprising neomycin sulfate. Thechromatogram for USP heparin was the base line case and served as astandard of reference for the heparin entities of Examples 1 and 2. Eachpeak in FIG. 5A represents a unique depolymerized oligosaccharidefragment characteristic of USP heparin. New peaks, as indicated by thevertical arrows in FIGS. 5B and C, represent novel oligosaccharidesunits distinct from USP heparin, and hence, allowed the identificationof heparin entities through these distinct signature peaks.

For the heparin entity of Example 1 comprising heparin and a corecomprising colistin sulfate, the chromatogram of FIG. 5B exhibits atleast 3 distinct peaks relative to the USP heparin chromatogram at15.581, 24.699, and 35.023 minutes (shown by vertical arrows).Structurally, these new peaks are related to the core molecule colistinsulfate utilized in the construction of the heparin entity. When theheparin entity was depolymerized with heparinase-1, new structurallydistinct polysaccharide units that contained the core molecule colistinsulfate were produced.

For the heparin entity of Example 2 comprising heparin and a corecomprising neomycin sulfate, the chromatogram of FIG. 5C exhibits atleast 3 distinct peaks relative to the USP heparin chromatogram at8.276, 25.386, and 34.867 minutes (shown by vertical arrows).Structurally, these new peaks are related to the core molecule neomycinsulfate utilized in the construction of the heparin entity. When theheparin entity was depolymerized with heparinase-1, new structurallydistinct polysaccharide units that contained the core molecule neomycinsulfate were produced.

Example 12

This example demonstrates a detection method for determining thecomposition of heparin entities immobilized on a surface usingoligosaccharide mapping of heparinase-1 depolymerized heparin entitieswith strong anion exchange-high performance liquid chromatography(SAX-HPLC).

Heparin comprising free-terminal aldehydes was immobilized onto disks ofePTFE/PEI according to Example 9. The heparin entity of Example 1 wasimmobilized onto disks of ePTFE/PEI according to Example 7. Samples ofapproximately 4 cm² of each disk were placed in individual tubes. Thesesamples were depolymerized to their constituent oligosaccharides by theaddition of 1 ml of acetate buffer (consisting of 50 mM sodium acetate,2.5 mM calcium acetate, pH 7.3) to each tube along with 60 μl ofheparinase-1 solution. The heparinase-1 solution comprised acetatebuffer (50 mM sodium acetate, 2.5 mM calcium acetate, pH 7.3) withheparinase-1 (EC 4.2.2.7, Sigma-Aldrich) at a concentration of 1.67IU/ml. Tubes were incubated at 30° C. for 18 hours, and liquid samplesof approximately 0.5 ml were taken and flash frozen at −85° C. forSAX-HPLC analysis as described in Example 11.

FIG. 6 shows the qualitative SAX-HPLC maps of surface depolymerized (A)heparin comprising free-terminal aldehydes immobilized on ePTFE and (B)a heparin entity constructed of heparin and a core of colistin sulfateimmobilized on ePTFE. The chromatogram for heparin comprisingfree-terminal aldehydes immobilized on ePTFE was the base line case andserved as a standard of reference for the heparin entity of Example 1.Each peak in FIG. 6A represents a unique oligosaccharide that ischaracteristic for heparin comprising free-terminal aldehydesimmobilized on ePTFE. New peaks, as indicated by arrows in FIG. 6B,represent additional oligosaccharides units distinct from heparincomprising free-terminal aldehydes immobilized on ePTFE, and hence,identify the heparin entity of heparin and a core of colistin sulfateimmobilized on ePTFE.

Example 13

This example describes the construction of a heparin entity comprisingheparin and a core comprising poly-L-lysine. This heparin entity doesnot contain free terminal aldehydes that can be used for attachment to asurface of a substrate. This heparin entity can be used for attachmentto a surface of a substrate through ionic bonding.

Poly-L-lysine hydrobromide with molecular weight of 1,000 to 5,000(0.1776 g, Sigma-Aldrich, St. Louis, Mo.) was dissolved in 300 ml of DIwater containing MES buffer (pH 4.7, BupH™ Thermo Scientific) and pHadjusted to 4.7. To this was added, 10 g USP heparin, 4 gN-hydroxysulfosuccinimide (sulfo-NHS), and 4 g of EDC hydrochloride. Thereaction was allowed to proceed at room temperature for 4 hours followedby dialysis overnight with 50,000 MWCO membrane (Spectra/Por®). Theretentate was transferred to 50 ml tubes, flash frozen, and lyophilizedto produce a fine powder. This powdered product was further used toimmobilize the construct of heparin and a core of poly-L-lysine on anePTFE sheet material through ionic bonding.

Example 14

The heparin entity of Example 13 was immobilized on the surface of thesubstrate ePTFE through ionic bonding.

Disks of ePTFE/PEI were prepared according to Example 7. The heparinentity of Example 13 containing a core of poly-L-lysine and no freeterminal aldehydes, was attached, via ionic bonding, to the PEI layer(s)by placing 5 1×1 cm square ePTFE samples of the construction in aheparin entity-containing sodium chloride salt solution (approximately0.247 g of heparin containing a core of poly-L-lysine containing noaldehydes, 0.16 g sodium citrate tri basic dehydrate, and 1.607 g NaCldissolved in 55 ml DI water, pH 3.9) and kept for one hundred and twentyminutes (120 min) at 60° C.

The samples were then rinsed in DI water for 15 min, borate buffersolution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1 LDI water, pH 9.0) for 20 min, and finally in DI water for 15 minfollowed by lyophilization of the entire construction to produce dryheparin bound to the ePTFE material. The presence and uniformity of theheparin containing a core of poly-L-lysine was determined by stainingsamples of the construction on both sides with toluidine blue. Thestaining produced an evenly stained surface indicating heparin waspresent and uniformly bound to the ePTFE material.

Example 15

This example demonstrates a detection method for determining theconjugation method for immobilizing heparin and heparin entities on asurface. Specifically, this example looks at the detection ofsurface-bonded unsaturated heparin fragments, or “nubs,” on the surfaceof immobilized heparin or heparin entities after heparinase-1depolymerization. Heparinase-1 depolymerization of heparin involves anenzymatic cleavage of heparin's non-reducing terminal uronic acid to a4,5-unsaturated derivative that can react with various detectionmolecules, such as a thiol-terbium fluorescent molecule. A negativecontrol of ionic bound heparin (Example 14) is included.

A thiol-terbium based florescent molecule was utilized. 5 gramshydroxypropyl β-cyclodextrin was dissolved into 50 ml DI-water, and0.03894 grams terbium tris(4-methylthio) benzoate [Tb(4MTB₃)] wasdissolved into 10 ml N,N-dimethylacetamide (DMAc). The Tb(4MTB₃)solution was then added drop wise into the hydroxypropyl β-cyclodextrinsolution, yielding a clear colorless solution. The solution was thenfiltered through a 0.22 μm Sterix filter cartridge before use.

Samples of 1 cm×1 cm ePTFE coated substrates of Example 14, and 1cm×1 cmsamples of the heparin entity of Example 1, comprising heparin and acore comprising colistin sulfate, immobilized according to Example 7,were depolymerized with heparinase-1 before reaction with thiol-terbium.For comparison, heparin end-point aldehyde (made according to U.S. Pat.No. 4,613,665) immobilized onto PEI-ePTFE substrates in accordance withExample 7, was also utilized; this produced a surface in which theheparin was immobilized by end-point attachment.

Samples were depolymerized with 100 units heparinase-1 (EC 4.2.2.7,Sigma-Aldrich) diluted in 1 ml of buffer (20 mM tris, 50 mM NaCl, 10 mMCaCl₂, 0.01% BSA, and pH 7.5) for 35 min on a shaker at roomtemperature. This resulted in small fragment “nubs” of surface-bondedunsaturated heparin fragments bound to the surface of the ePTFEsubstrate. The samples were then rinsed in DI water for 15 min, boratebuffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolvedin 1 L DI water, pH 9.0) for 20 min, and stored in DI-water until usedfor final analysis. Fluorescence labeling of samples, through aMichael-like addition of the thiol-terbium compound to the unsaturatedheparin fragment bound to the surface of the ePTFE substrate, wasperformed by placing each sample into vials containingTb(4MTB₃)/hydroxypropyl β-cyclodextrin solution, purged with nitrogenfor 1 minute, capped, and incubated overnight at 70° C. Samples wereremoved from vials, rinsed with 10 wt % hydroxypropyl β-cyclodextrin inDI-water, and placed on a glass microscope slide for imaging.

Imaging of samples was performed with a Nikon E-6000 microscope using anOcean Optics Deuterium short-wavelength excitation source at an obliqueangle. Both white light and UV excitation fluorescence images were takenusing a FITC filter cube. All samples were maintained in a wet stateduring imaging to minimize background scattering, and imaged with ablack and white camera. Samples excited with UV light were imaged, andgreen tinting was artificially added to the image for visualizationpurposes.

Distinct UV fluorescence, and hence the detection of surface-bondedunsaturated heparin fragments bound to the surface of the ePTFEsubstrate (“nubs”), was noted for the end-point aldehyde heparin andheparin entity comprising heparin and a core comprising colistin sulfatesamples. An absence of UV fluorescence was noted for the macromolecularconstruct of ionically bound heparin and poly-L-lysine containing noaldehydes.

Example 16

This example describes the construction and utilization of an embodimentof the present invention in which high heparin anti-thrombin III (ATIII)binding is present for a heparin entity comprising heparin and a corecomprising an amine-containing fluoropolymer. This heparin entitycontains free terminal aldehydes that can be used for attachment to asurface of a substrate.

The amine-containing fluoropolymer was prepared using the followingconditions. A copolymer comprising a mole ratio of 20:80tetrafluoroethylene and vinyl acetate was prepared. To a nitrogen purged1 L pressure reactor under vacuum were added 500 g DI water, 2 g of 20%aqueous ammonium perfluorooctanoate, 30 ml of distilled vinyl acetate,10 g of n-butanol, and 0.2 g of ammonium persulfate. Tetrafluoroethylenemonomer was then fed into the reactor until the reactor pressure reached1500 KPa. The mixture was stirred and heated to 50° C. When a pressuredrop was observed, 25 ml of vinyl acetate was slowly fed into thereactor. The reaction was stopped when the pressure dropped another 150KPa after vinyl acetate addition. The copolymer was obtained fromfreeze-thaw coagulation of the latex emulsion and cleaned withmethanol/water extraction. The copolymer then was hydrolyzed. To a 50 mlround bottle flask were add 0.5 g of the copolymer, 10 ml methanol and0.46 g NaOH in 2 ml DI water. The mixture was stirred and heated to 60°C. for 5 hrs. The reaction mixture was then acidified to pH 4 andprecipitated in DI water. The hydrolyzed copolymer was then acetalized.The hydrolyzed copolymer was dissolved in methanol at 2.5% w/v. To 50 gof this solution was added 33 ml of DI water with vortexing to produce ahomogeneous solution. To this solution was added 0.153 g ofaminobutyraldehyde dimethyl acetal, and 0.120 ml of a 37% HCl solution.The solution was reacted with stirring under nitrogen, 80° C., for 48hrs. Sodium hydroxide from a 1M solution was added drop wise to a pH ofabout 9.0. The resulting copolymer of poly(tetrafluoroethylene-co-vinylalcohol-co-vinyl[aminobutyraldehyde acetal]) (TFE-VOH-AcAm) wasrecovered by precipitation into copious DI water. The precipitate wasfiltered, redissolved into methanol, and reprecipitated into copious DIwater for two more cycles. The final product was dried under vacuum at60° C. for 3 hrs. FTIR and carbon NMR confirmed a polymer structure ofpoly(tetrafluoroethylene-co-vinyl alcohol-co-vinyl[aminobutyraldehydeacetal]).

48 mg of aldehyde-modified-heparin (made according to U.S. Pat. No.4,613,665) was dissolved in 30 ml of DI water. To this solution wasadded 86 μl of 2.5% sodium cyanoborohydride solution (Aldrich) and thepH was adjusted to 3.8 with HCl. Separately, the TFE-VOH-AcAm copolymerwas dissolved in superheated methanol at 2.5% w/v and then cooled toroom temperature. To 20 ml of the TFE-VOH-AcAm solution was added 13 mlof the heparin solution drop wise, to produce a slightly milky emulsion.The emulsion was maintained at 60° C. for 2.5 hrs and then at roomtemperature for an additional 2 hrs. The emulsion was dialyzed againstDI water using a 50KDa membrane (SpectraPor) for 18 hrs, flash frozen at−80° C. and then lyophilized to a powder. 10 mg of the powder wassuspended in 2.5 ml of ice cold DI water supplemented with 0.1 mg sodiumnitrite (Sigma) and 20 μl of acetic acid (Baker). After reacting for 2hrs at 0° C., the suspension was dialyzed against DI water using a 10KDa membrane (SpectaPor) for 18 hrs, flash frozen at −80° C. and thenlyophilized.

Example 17

This example describes the construction and utilization of an embodimentof the present invention in which high heparin ATIII binding is presentfor heparin entity comprising heparin and a core comprising anamine-containing fluoropolymer. This heparin entity contains aldehydesalong the length of the heparin component that can be used forattachment to a surface of a substrate.

48 mg of aldehyde-modified-heparin (made according to U.S. Pat. No.4,613,665) was dissolved in 30 ml of DI water. To this solution wasadded 86 μl of 2.5% sodium cyanoborohydride solution (Aldrich), and thepH adjusted to 3.8 with HCl. Separately, the TFE-VOH-AcAm copolymer ofExample 16 was dissolved in superheated methanol at 2.5% w/v and thencooled to room temperature. To 20 ml of the TFE-VOH-AcAm solution wasadded 13 ml of the heparin solution drop wise to produce a slightlymilky emulsion. The emulsion was maintained at 60° C. for 2.5 hrs andthen at room temperature for an additional 2 hrs. The emulsion wasdialyzed against DI water using a 50 KDa membrane (SpectraPor) for 18hrs, flash frozen at −80° C. and then lyophilized to a powder.

A solution was prepared containing 100 ml DI water, 0.82 g sodiumacetate, and 0.128 g sodium periodate (ICN). To 12 ml of this solutionwas suspended 12 mg of the powder. After reacting for 30 min in thedark, 1.2 ml of glycerol was added to quench the reaction, thesuspension was dialyzed against DI water using a 10 KDa membrane(SpectaPor) for 18 hrs, flash frozen at −80° C. and then lyophilized.

Example 18

The heparin entities of Examples 16 and 17, comprising heparin and acore comprising amine-containing fluoropolymer, were immobilized ontothe ePTFE/PEI substrates, following the method described in Example 7,except that the samples were not exposed to EtO. ATIII binding activitywas measured following the method described in Example 7.

TABLE 3 ATIII binding activity Example # Attachment type pmol/cm2 16Free terminal aldehyde 106 17 Aldehyde along chain length 66 (loopattachment)

Example 19

This example describes the construction and utilization of an embodimentof the present invention in which high heparin ATIII binding is presentfor heparin entity comprising heparin and a core comprising an aminecontaining fluoropolymer. This heparin entity contains free terminalaldehydes that can be used for attachment to a surface of a substrate.

The amine containing fluoropolymer was prepared using the followingconditions. A 4 L reactor was charged with 2 L of t-butanol. 50 g oftetrafluoroethylene (TFE), 200 g of perfluoromethylvinylether (PMVE) and100 g of N-vinyl formamide (NFA) were added, along with 0.4 g ofdiisopropyl peroxydicarbonate as initiator. The solution was stirred ata speed of 800 rpm at 70° C. for 3 hrs. The precipitate was removed fromthe reactor, air-dried for 2 hrs, and dried at 40° C. under vacuum for24 hrs. Proton and fluorine NMR analysis confirmed a TFE-PMVE-NFApolymer composition of 46 weight % NFA, 27 weight % PTFE and 27 weight %PMVE. This polymer was soluble in methanol and swelled in water.

25 g of the TFE-PMVE-NFA polymer was dispersed in 100 mL of DI water.The mixture was heated to 70° C., and 30 mL of 37% HCl was slowly added.The solution was kept at 90° C. for 4 hrs. Hydrolyzed polymer wasrecovered from acetone precipitation, air-dried for 2 hrs, and dried at40° C. under vacuum for 24 hrs. FTIR analysis confirmed hydrolysis ofthe vinyl formamide groups to vinyl amine (VA) groups. The TFE-PMVE-VApolymer was water soluble.

In a vial, 2 g of USP Heparin was dissolved in 50 mL of 0.1M MES buffer,containing 0.8 g of EDC and 0.8 g sulfo-NHS. In a second vial, a secondsolution was prepared consisting of 1 g of TFE-PMVE-VA polymer and 30 mLof 0.1M MES buffer. The heparin solution was added drop wise into thepolymer solution over 4 hrs at room temperature, and pH maintained at4.7 with 1.0N NaOH. The reaction was kept overnight at room temperature.The solution was dialyzed in DI water for two days with 10,000 MWCOmembrane (Spectra/Por®). The retentate was concentrated with rotaryevaporation.

0.01 g NaNO2, 100 mL of DI water, and 2 mL of acetic acid were added tothe retentate. The reaction proceeded at 0° C. for 2 hrs, followed bydialysis against DI water for two days, flash frozen at −80° C. and thenlyophilized.

It will be apparent to those skilled in the art that variousmodifications and variation can be made in the present invention withoutdeparting from the spirit or scope of the invention. Thus, it isintended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

1. A heparin entity comprising: at least one heparin molecule and atleast one core molecule such that when said heparin entity is bound ontoa substrate via said at least one heparin molecule, said heparin entityis heparinase sensitive.
 2. The heparin entity of claim 1, wherein saidsubstrate is selected from the group consisting of polyethylene,polyurethane, silicone, polyamide-containing polymers, polypropylene,polytetrafluoroethylene, expanded-polytetrafluoroethylene andbiocompatible metals.
 3. The heparin entity of claim 1, wherein saidsubstrate is expanded-polytetrafluoroethylene.
 4. The heparin entity ofclaim 2, wherein said biocompatible metal is Nitinol.
 5. The heparinentity of claim 1, wherein said substrate is a component of a medicaldevice.
 6. The heparin entity of claim 5, wherein said medical device isselected from the group consisting of grafts, vascular grafts, stents,stent-grafts, bifurcated grafts, bifurcated stents, bifurcatedstent-grafts, patches, plugs, drug delivery devices, catheters, cardiacleads, balloons and indwelling filters.
 7. The heparin entity of claim6, wherein said stents can be used in cardiac, peripheral orneurological applications.
 8. The heparin entity of claim 6, whereinsaid stent-grafts can be used in cardiac, peripheral or neurologicalapplications.
 9. The heparin entity of claim 1, wherein said coremolecule is either cyclic, linear, branched, dendritic, T, Y or starshaped.
 10. The heparin entity of claim 1, wherein said core molecule isselected from the group consisting of proteins, polypeptides,hydrocarbons, polysaccharides, aminoglycosides, and polymers.
 11. Theheparin entity of claim 10, wherein said protein is selected from thegroup consisting of albumin, colistin and polylysine.
 12. The heparinentity of claim 10, wherein said polysaccharide is selected from thegroup consisting of cyclodextrin, cellulose, and chitosan.
 13. Theheparin entity of claim 10, wherein said polymer is selected from thegroup consisting of polyethylene glycol (PEG) and co-polymers oftetrafluoroethylene.
 14. The heparin entity of claim 1, wherein saidheparin is derived from bovine or porcine sources.
 15. The heparinentity of claim 1, wherein after heparinase treatment heparin orfragments thereof will not be detected on said substrate.
 16. Themedical substrate of claim 1, wherein after heparinase treatment heparinor fragments thereof will be detect at a significantly lower level thanbefore heparinase treatment.
 17. The heparin entity of claim 15, whereinheparin or fragments thereof is detected by a label that binds toheparin or fragments thereof.
 18. The heparin entity of claim 17,wherein said label that binds to heparin or fragments thereof isselected from the group consisting of dyes, antibodies, and proteins.19. The heparin entity of claim 18, wherein said dye is toluidine blue.20. The heparin entity of claim 1, wherein after heparinase treatment aninsignificant amount of toluidine blue will bind to residual heparin orfragments thereof but will not be visually detected on said substrate.21. The heparin entity of claim 1, wherein after heparinase treatment aninsignificant amount of toluidine blue will bind to residual heparin orfragments thereof, and wherein detector readings will be aboutbackground levels or be insignificantly different from background levelswhen compared to a substrate without heparin entities.
 22. The heparinentity of claim 1, wherein said heparin entity is bound onto a substratevia at least one heparin molecule and wherein said bound heparinmolecule is attached to said substrate via end point attachment.
 23. Theheparin entity of claim 1, wherein said heparin entity is bound onto asubstrate via at least one heparin molecule and wherein said boundheparin molecule is attached to said substrate via loop attachment. 24.The heparin entity of claim 1, wherein said heparin entity is bound ontoa substrate via at least one heparin molecule and wherein said boundheparin molecule is attached to said substrate via end point aldehyde.25. The heparin entity of claim 1, wherein said heparin entity is boundonto a substrate via at least one heparin molecule and wherein saidbound heparin molecule is attached to said substrate via aldehydes alongthe length said heparin.
 26. An ATIII binding entity comprising; a coremolecule, a polysaccharide chain attached to the core molecule, and afree terminal aldehyde moiety on the polysaccharide chain.
 27. The ATIIIbinding entity of claim 26, wherein said polysaccharide chain isheparin.
 28. The ATIII binding entity of claim 26, wherein said coremolecule is selected from the group consisting of a protein, ahydrocarbon, an aminoglycoside, a polysaccharide and a polymer.
 29. TheATIII binding entity of claim 28, wherein said protein is selected fromthe group consisting of albumin, colistin, and polylysine.
 30. The ATIIIbinding entity of claim 28, wherein said polysaccharide is selected fromthe group consisting of cyclodextrin, cellulose, and chitosan.
 31. TheATIII binding entity of claim 28, wherein said polymer is selected fromthe group consisting of polyethylene glycol (PEG) and co-polymers oftetrafluoroethylene.
 32. The ATIII binding entity of claim 27, whereinsaid heparin is derived from bovine or porcine sources.
 33. The ATIIIbinding entity of claim 27, wherein said heparin is bound onto the coremolecule via end point attachment.
 34. The ATIII binding entity of claim27, wherein said heparin is bound onto a substrate via end pointattachment.
 35. The ATIII binding entity of claim 34, wherein saidsubstrate is selected from the group consisting of polyethylene,polyurethane, silicone, polyamide-containing polymers, andpolypropylene, polytetrafluoroethylene, expanded-polytetrafluoroethyleneand biocompatible metals.
 36. The ATIII binding entity of claim 35,wherein said substrate is expanded-polytetrafluoroethylene.
 37. TheATIII binding entity of claim 35, wherein said biocompatible metal isNitinol.
 38. The ATIII binding entity of claim 34, wherein saidsubstrate is a component of a medical device.
 39. The heparin entity ofclaim 38, wherein said medical device is selected from the groupconsisting of grafts, vascular grafts, stents, stent-grafts, bifurcatedgrafts, bifurcated stents, bifurcated stent-grafts, patches, plugs, drugdelivery devices, catheters and cardiac leads.
 40. The heparin entity ofclaim 39, wherein said stents can be used in cardiac, peripheral orneurological applications.